Pseudo-thermosetting neutralized chitosan composition forming a hydrogel and a process for producing the same

ABSTRACT

The composition disclosed is a pseudo-thermosetting neutralized chitosan composition, neutralized with an hydroxylated base, forming a phosphate-free transparent hydrogel at a temperature higher than 5° C. Said composition contains a homogeneously reacetylated chitosan derived from a chitosan having a deacetylation degree of 80-90%, having a molecular weight of not smaller than 200 kDa and a deacetylation degree of 30-60%, and may further contain a diol. Said composition may be used as a drug delivery system.

FIELD OF THE INVENTION

The present invention relates to a pseudo-thermosetting neutralizedchitosan composition forming a phosphate-free, transparent hydrogel at atemperature higher than 5° C., and to a process for producing the same.

Still further, the present invention relates to a process for producinga homogeneously reacetylated chitosan, to a homogeneously reacetylatedchitosan obtained by said process for use in the preparation of apseudo-thermosetting neutralized chitosan composition forming aphosphate-free, transparent hydrogel at a temperature higher than 5° C.,and to the use of a pseudo-thermosetting neutralized chitosancomposition as a drug delivery system.

BACKGROUND OF THE INVENTION

Hydrogels, which may be defined as macromolecular networks swollen inwater or biological fluids, are known for various biomedicalapplications.

Further, hydrogels exhibiting the specific property of having theirviscosity increasing when the temperature increased, also called“thermosensitive hydrogels”, were proved to have a facilitatedapplication combined with an increased residence time at the site ofapplication and therefore were found advantageous as drug deliverysystem.

As known from O. Felt et al. in The Encyclopedia of Controlled DrugDelivery, 1999, said thermosensitive hydrogels may be basedadvantageously on polymers of natural origin, for example on chitosanwhich is a commercially available inexpensive polymer derived fromchitin, the second most abundant polysaccharide after cellulose.

Chitosan is known as a chitin derivative obtained by partial tosubstantial alkaline N-deacetylation of chitin also namedpoly(N-acetyl-D-glucosamine), which is a naturally occurring biopolymer,found in hard shells of marine living animals such as fishes,crustaceous, shrimps, crabs, etc., or synthesized by natural organismssuch as zygomycete, fungi, etc.

Chitosan contains free amine (—NH₂) groups and may be characterized asto the proportion of N-acetyl-D-glucosamine units and D-glucosamineunits, and such is expressed as the degree of deacetylation (DD) of thefully acetylated polymer chitin.

Parameters of chitosan influencing important properties such assolubility and viscosity are the degree of deacetylation (DD) which maybe understood as representing the percentage of deacetylated monomers,and the molecular weight (MW).

Chitosan is known to be biodegradable, biocompatible, bioadhesive,bacteriostatic, and further to promote wound-healing, drug absorption,and tissue reconstruction.

Due to its above mentioned intrinsic properties, chitosan is known tohave numerous cosmetic and pharmaceutical activities, and has been alsowidely explored for various applications through gels.

Therefore, considering the advantageous properties of chitosan, there isa continuous need to improve the properties of known thermosensitivechitosan hydrogels which are still considered as very promising for awider range of biomedical applications.

WO-A-99/07416 (Biosynthec) discloses a pH-dependenttemperature-controlled chitosan hydrogel which has thermosensitiveproperties at neutral pH such that it has low viscosity in the cold butgels at body temperature.

This thermosensitive chitosan hydrogel is prepared by neutralizing acommercial chitosan having a deacetylation degree of about 80% withmono-phosphate dibasic salts of polyols or sugars exemplified inparticular by β-glycerophosphate (β-GP).

Addition of β-GP to chitosan allows to increase the pH up to 7 withoutchitosan precipitation and to form a hydrogel on a temperature dependantway, i.e. the higher is the temperature, the faster is the gelationprocess.

Said hydrogels are advantageous in that they contain biocompatiblecomponents and a high percentage of water, in that they have aphysiological pH and in that no heat nor product is released duringgelation.

As also reported by A. Chenite et al. in Carbohydr. Polym. 46, 39-47(2001), in relation with chitosan/β-GP pseudo-thermosetting hydrogels,adding β-GP for neutralizing high DD chitosan which are known toprecipitate above pH 6.2 allows to prevent precipitation of said high DDchitosan.

However, presence of β-GP in the hydrogel leads to the followingdisadvantages.

β-GP is a negatively charged entity that can react with a positivelycharged bioactive component, leading to its precipitation or to thedisturbance of its liberation from the hydrogel.

Therefore, presence of β-GP renders chitosan/β-GP hydrogelsinappropriate for use with numerous drugs.

Further, the modulation of the properties of this hydrogel, such asgelation time and viscosity, depends on the concentration of β-GP and istherefore limited by the solubility of β-GP.

In particular, a high concentration of β-GP is required to have a lowgelation time avoiding the rapid elimination of the hydrogel after itsadministration.

However, a high concentration of β-GP also decreases the viscosity ofthe hydrogel.

Therefore, the gelation time has to be balanced with the consistency ofthe hydrogel, and it is not possible to obtain gels that have both a lowgelation time and a high viscosity, which would be a desirablecombination of characteristics.

Also, a too high concentration of β-GP may induce the precipitation ofthe hydrogel at its administration site.

Further, said thermosensitive chitosan/β-GP hydrogels were found to beturbid, thus rendering their use inappropriate for particularapplications such as ocular or topic administrations.

On the basis of these facts, the present inventors have continued theirresearches to overcome the disadvantages of the known thermosensitivechitosan/β-GP hydrogels and have surprisingly found that by using areacetylated chitosan having a deacetylation degree of 30-60%,neutralization of chitosan to form thermosensitive hydrogel may be madeby addition of NaOH or any other hydroxylated base instead ofβ-glycero-phosphate, and further that if reacetylation of chitosan to DD30-60% is made in homogeneous conditions, a transparent chitosanhydrogel is obtained.

The present invention has been achieved on the basis of these results.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides apseudo-thermosetting neutralized chitosan composition, which comprises0.1 to 2.0 wt/v %, preferably 0.5 to 1 wt/v %, based on the totalcomposition, of a homogeneously reacetylated chitosan derived from achitosan having a deacetylation degree of 80-90%, having a molecularweight of not smaller than 200 kDa, preferably not smaller than 600 kDa,and a deacetylation degree of 30-60%, preferably 45 to 55%, neutralizedwith an hydroxylated base, wherein said composition forms aphosphate-free transparent hydrogel at a temperature higher than 5° C.In a preferred embodiment, the pseudo-thermosetting neutralized chitosancomposition further comprises a diol having a distance of at least 4.7 Åbetween its hydroxyl groups, preferably 1,3-propanediol.

According to a second aspect, the present invention provides a processfor producing a homogeneously reacetylated chitosan having a molecularweight of not smaller than 200 kDa and a deacetylation degree of 30-60%which comprises the steps of: a) filtrating a chitosan having amolecular weight of not smaller than 200 kDa and a deacetylation degreeof 80 to 90% dissolved in an acidic medium to eliminate insolubleparticles; b) precipitating chitosan contained in the filtrated acidicsolution obtained in step a) to obtain chitosan free of insolubleparticles; c) preparing a cooled acidic solution of the chitosan free ofinsoluble particles obtained in step b) at a temperature lower than 5°C. to obtain a cooled acidic solution of chitosan free of insolubleparticles; d) preparing a cooled acetic anhydride solution containing apredetermined amount of acetic anhydride in methanol at a temperaturelower than 5° C.; e) reacetylating chitosan by adding, under homogeneousconditions, the cooled acetic anhydride solution of step d) to thecooled solution of chitosan prepared in step c) to provide a crudehomogeneously reacetylated chitosan having a deacetylation degree of30-60% ; f) treating said crude chitosan obtained in step e) toeliminate salts produced during reacetylation and insoluble particles ofchitosan to obtain a homogeneously reacetylated chitosan having adeacetylation degree of 30-60%. In a preferred embodiment, the treatingstep f) includes the steps of: f-1) dialyzing chitosan obtained in stepe) to eliminate salts produced during reacetylation in order to obtain ahomogeneously reacetylated chitosan solution; f-2) filtrating thechitosan solution obtained in step f-1) to eliminate insoluble particlesof chitosan; f-3) precipitating chitosan contained in the filtratedsolution obtained in step f-2) and then drying chitosan to obtain ahomogeneously reacetylated chitosan having a deacetylation degree of30-60%. Preferably, the precipitating step f-3) includes addition of amixture of NH₄OH/methanol.

According to a third aspect, the present invention provides ahomogeneously reacetylated chitosan having a molecular weight of notsmaller than 200 kDa and a deacetylation degree of 30-60% obtained bythe process according to the second aspect of the present invention, foruse in the preparation of a pseudo-thermosetting neutralized chitosancomposition forming a phosphate-free transparent hydrogel at atemperature higher than 5° C.

According to a fourth aspect, the present invention provides a processfor producing a pseudo-thermosetting neutralized chitosan compositionforming a phosphate-free, transparent hydrogel at a temperature higherthan 5° C., which comprises the steps of: g) solubilizing ahomogeneously reacetylated chitosan derived from a chitosan having adeacetylation degree of 80-90%, having a molecular weight of not smallerthan 200 kDa and a deacetylation degree of 30-60%, in an aqueous HClmedium and cooling said acidic chitosan solution at a temperature lowerthan 5° C.; h) neutralizing the cooled chitosan solution obtained instep g) by adding an aqueous hydroxylated base, preferably NaOH,previously cooled at a temperature lower than 5° C. to the cooledchitosan solution until the cooled solution of chitosan exhibits a pH of6.8 to 7.2, preferably a pH of 7; i) optionally, increasing thetemperature of the neutral cooled solution of chitosan obtained in steph) at a temperature higher than 5° C. in order to inducepseudo-thermogelation. In a preferred embodiment, the process furthercomprises a step of sterilizing chitosan before the step g) ofsolubilization. In a still preferred embodiment, the process furthercomprises the step of adding an appropriate amount of a diol having adistance of at least 4.7 Å between the hydroxyl groups, preferably1,3-propanediol, before, during or after solubilization step g), orbefore, during or after the neutralization step h) to increase theconsistency of the hydrogel to the required degree of consistency of thehydrogel. In a still further preferred embodiment, the homogeneouslyreacetylated chitosan solubilized in step g) is obtained by the processaccording to the second aspect of the present invention.

According to a fifth aspect, the present invention provides aphosphate-free transparent pseudo-thermosetting chitosan hydrogelobtained by the process according to the fourth aspect of the presentinvention.

According to a sixth aspect, the present invention provides a use of ahomogeneously reacetylated chitosan having a deacetylation degree of30-60% and a molecular weight of not smaller than 200 kDa obtained bythe process according to the second aspect of the present invention, forthe preparation of a phosphate-free, transparent, pseudo-thermosettingchitosan hydrogel.

According to a seventh aspect, the present invention provides a use of apseudo-thermosetting neutralized chitosan composition according to thefirst aspect of the present invention, as a drug delivery system.

According to the present invention, using a reacetylated chitosan havinga deacetylation degree of 30-60% allows advantageously to use NaOH orany other hydroxylated base to neutralize the chitosan instead of usingβ-glycero-phosphate and therefore allows to obtain a phosphate-freechitosan hydrogel.

Further, according to the present invention, using a reacetylatedchitosan which has been reacetylated in homogeneous conditions to adeacetylation degree of 30-60% allows advantageously to obtain atransparent chitosan hydrogel.

Still further, according to the present invention, addition of diolshaving a minimal distance of 4.7 Å between the hydroxyl groups allowsadvantageously to modulate the viscoelastic properties of the hydrogel.

Other advantages of the present invention will appear in the followingdescription.

The present invention will be now described in a more detailed manner.

BRIEF DESCRIPTION OF THE FIGURE

The Figure shows the elastic modulus (G′) of two phosphate-free,transparent chitosan hydrogels of the present invention obtained inExamples 6 and 7, and of a comparative phosphate-containing chitosanhydrogel obtained in Example 10 (Comparative) as a function of time whentemperature increases from 4 to 37° C.

DETAILED DESCRIPTION OF THE INVENTION

It is to be noted that in the present description and claims, theexpression “pseudo-thermosetting” in connection with the composition ofthe present invention means that temperature does not induce thegelation of the composition but acts rather as a catalyst whichdramatically shortens the gelation time when risen.

It is also to be noted that in the present description and claims, theterm “neutralized” means a pH of 6.8-7.2.

According to the present invention, the pseudo-thermosetting neutralizedchitosan composition forming a phosphate-free transparent hydrogel at atemperature higher than 5° C. comprises a homogeneously reacetylatedchitosan neutralized with an hydroxylated base.

The average molecular weight (MW) of the homogeneously reacetylatedchitosan used in the present invention must be not smaller than 200 kDa.

Molecular weight of chitosan may be easily determined by size exclusionchromatography as reported for example by O. Felt, P. Furrer, J. M.Mayer, B. Plazonnet, P. Burri and R. Gurny in Int. J. Pharm. 180,185-193 (1999).

Chitosan of molecular weights lower than 200 kDa is not appropriate foruse in the present invention because it would not allow the formation ofa pseudo-thermosetting composition forming a firm hydrogel.

Preferably, the homogeneously reacetylated chitosan used in the presentinvention has a molecular weight of not lower than 600 kDa.

The upper limit of MW is determined by the ease of administration, whichdepends on the chosen application.

Homogeneously reacetylated chitosan used in the present invention musthave a deacetylation degree of 30-60% which means that the chitosancomprises 30 to 60% of D-glucosamine units and 70 to 40% of neutralN-acetyl-D-glycosamine units, respectively.

The deacetylation degree of chitosan may be determined by aspectrophotometric method such as described in the literature by R. A.Muzarelli and R. Richefti in Carbohydr. Polym. 5, 461-472, 1985 or R. A.Muzarelli and R. Richetti in “Chitin in Nature and Technology”, PlenumPress 385-388, 1986. Briefly, in the latter method for example, chitosanis solubilized in 1% acetic acid and the DD is determined by measuringits content of N-acetyl-glucosamine by UV at λ 200, 201, 202, 203 and204 nm using N-acetyl-D-glucosamine solutions as standards.

If deacetylation degree of chitosan is lower than 30%, the chitosanbecomes a polymer close to chitin that is insoluble in acidic conditionsand consequently not usable in the present invention.

If deacetylation degree of chitosan is higher than 60%, the chitosandoes not allow the preparation of a composition forming a phosphate-freetransparent hydrogel.

In the present invention, in addition to the proportion of acetylatedand deacetylated monomers of chitosan represented by its degree ofdeacetylation, the homogeneous distribution mode of these monomers is anessential criteria to get transparent and phosphate-free hydrogels.

In order to obtain such homogeneous distribution mode of acetylated anddeacetylated monomers, the chitosan used for preparing thepseudo-thermosetting composition forming a hydrogel must be a chitosanderived from a chitosan having a deacetylation degree of 80-90% whichhas been homogeneously reacetylated to a deacetylation degree of 30-60%in conditions allowing a random distribution of acetylated anddeacetylated monomers.

If the chitosan is reacetylated in a non-homogeneous manner, thechitosan does not allow the preparation of a transparent hydrogel.

Preferably, the homogeneously reacetylated chitosan used in the presentinvention has a deacetylation degree of 45 to 55%.

An homogeneously reacetylated chitosan having a molecular weight of notsmaller than 200 kDa and a deacetylation degree of 30-60% for use in thepreparation of a pseudo-thermosetting neutralized chitosan compositionforming a phosphate-free, transparent hydrogel of the present inventionmay be prepared from a commercially available chitosan having amolecular weight of not smaller than 200 kDa and a deacetylation degreeof 80 to 90% according to a new process forming part of the presentinvention which is based on the reacetylation method of chitosan withacetic anhydride described by Hirano (Hirano S. et al., Carbohydr. Res.47, 315-320 (1976).

In step a) of the process of the present invention for preparing ahomogeneously reacetylated chitosan, a commercial chitosan having amolecular weight not smaller than 200 kDa and a deacetylation degree of80 to 90%, dissolved in an acidic medium, is filtrated to eliminateinsoluble particles.

An example of commercial chitosan which may be used in the process ofthe present invention is a chitosan of pharmaceutical grade and high MWobtained from Aldrich Chemical, Milwaukee, USA, having a MW of 1′100 kDaas determined by size exclusion chromatographic method reported by O.Felt, et al. in Int. J. Pharm. 180, 185-193 (1999) and a deacetylationdegree DD of 83.2% as measured by UV method reported by R. A. Muzarelliet al. in “Chitin in Nature and Technology”, Plenum Press, New York,385-388, (1986).

However, any commercial chitosan having a deacetylation degree of 80 to90% and a molecular weight not smaller than 200 kDa may be used.

The acidic medium used for dissolving commercial chitosan may be forexample 10% acetic acid and the acidic solution of chitosan obtainedafter solubilization of chitosan may be then diluted with an alcohol,for example methanol.

The acidic solution containing chitosan must be filtered to eliminateinsoluble particles because presence of insoluble particles does notallow a subsequent homogeneous reacetylation.

Said filtration may be made for example in two times, first through a100 μm filter and then through a 5 μm filter, but any other mesh sizesmay be used.

Then, in step b) of the process of the present invention for preparing ahomogeneously reacetylated chitosan, chitosan contained in the filtratedacidic solution is precipitated, for example by adding a 0.2 MNH₄OH/methanol (50/50) solution.

After precipitation, the chitosan may be further washed, for examplewith methanol, and then dried according to conventional methods, forexample in the presence of Silicagel, under vacuum, at room temperatureand protected from light.

In step c) of the process of the present invention for preparing ahomogeneously reacetylated chitosan, chitosan obtained in step b) isfurther dissolved in an acidic medium, for example 10% acetic acid, andthe acidic solution of chitosan obtained may be diluted, for examplewith methanol. Said acidic solution is then cooled at a temperaturelower than 5° C., thus obtaining a cooled acidic solution of chitosanfree of insoluble particles.

According to step d) of the process of the present invention forpreparing a homogeneously reacetylated chitosan, it is preparedseparately an acetic anhydride solution containing a predeterminedamount of cooled acetic anhydride in cooled methanol (for example in anice-bath), so this solution is at a temperature lower than 5° C.

According to step e) of the process of the present invention forpreparing a homogeneously reacetylated chitosan, chitosan isreacetylated by adding dropwise, under homogeneous conditions, thecooled acetic anhydride solution containing a predetermined amount ofacetic anhydride to the cooled acidic solution containing chitosan inorder to provide a chitosan having a deacetylation degree of 30-60%.

A temperature of the acetic anhydride solution and/or of the cooledchitosan solution higher than 5° C. would not allow a homogeneousreacetylation.

The amount of acetic anhydride contained in the acetic anhydridesolution will depend on the amount of the chitosan to reacetylate, onthe degree of deacetylation of the commercial starting chitosan and onthe degree of deacetylation that is intended to obtain.

Therefore, said amount of acetic anhydride contained in the aceticanhydride solution will be determined from case to case.

Preferably, said amount of acetic anhydride will be predetermined toobtain a degree of deacetylation of the reacetylated chitosan of 45 to55%.

Homogeneous conditions during the addition step e) is an essentialfeature of this process, and they may be obtained by providing a faststirring during the addition, for example with a stirring propeller.

An inadequate stirring, for example with a magnetic stirrer, does notallow a homogeneous reacetylation of chitosan and therefore, thechitosan obtained would not allow the preparation of apseudo-thermosetting composition forming a transparent hydrogel.

After addition of cooled acetic anhydride solution to the cooledsolution containing chitosan, the solution containing homogeneouslyreacetylated chitosan may be kept further under stirring at atemperature lower than 5° C. to ensure complete reaction and then may bekept at room temperature where it turns into a gel.

According to step f) of the process of the present invention forpreparing a homogeneously reacetylated chitosan, the crude homogeneouslyreacetylated chitosan obtained after the reacetylation step e) must betreated to eliminate salts produced during reacetylation and to furthereliminate insoluble particles.

Said treatment may be preferably carried out by dialyzing homogeneouslyreacetylated chitosan, for example in the gel form which may be obtainedafter step e), against deionized water for a sufficient time toeliminate the salts produced during reacetylation, thus leading to achitosan viscous solution, then by filtering the chitosan viscoussolution through a 100 μm filter or other size filter in order toeliminate insoluble particles of chitosan.

Homogeneously reacetylated chitosan having a deacetylation degree of30-60% for use in the preparation of a phosphate-free, transparentchitosan hydrogel may be recovered for example after precipitation byaddition of 0.2 M NH₄OH/methanol (50/50), washing, for example, withmethanol, and drying, for example in the presence of Silicagel, undervacuum, at room temperature and protected from light.

The pseudo-thermosetting neutralized chitosan composition forming aphosphate-free, transparent hydrogel of the present invention isprepared according to a new process forming part of the presentinvention, using a homogeneously reacetylated chitosan preparedaccording to steps a) to f) of the process for preparing a homogeneouslyreacetylated chitosan forming part of the present invention.

If the chitosan is not homogeneously reacetylated, the obtained hydrogelwill be turbid or chitosan could precipitate during neutralization.

Typically hydrogels are prepared with chitosan concentration rangingfrom 0.1 to 2.0 wt/v %, based on the total composition.

Lower concentrations of chitosan do not allow the formation of ahydrogel and higher concentrations induce the formation of a too firmhydrogel that is not useable.

Preferably, chitosan concentration ranges from 0.5 to 1.0 wt/v %, basedon the total composition.

According to step g) of process of the present invention for preparingthe composition forming a hydrogel, homogeneously reacetylated chitosanhaving a molecular weight of not smaller than 200 kDa, preferably notsmaller than 600 kDa and a deacetylation degree of 30-60%, preferably45-55%, is solubilized in an aqueous HCl medium and after completedissolution of chitosan, the temperature of the chitosan solution iscooled down to a temperature lower than 5° C., for example in anice-bath.

A higher temperature would induce the precipitation of chitosan duringits neutralization.

Then, according to step h) of the process of the present invention forpreparing the composition forming a hydrogel, the pH of the chitosansolution is neutralized until pH 6.8-7.2, preferably pH 7 by addingdropwise, under stirring at a temperature lower than 5° C., the requiredamount of an aqueous solution containing a hydroxylated base previouslycooled at a temperature lower than 5° C.

A higher pH would induce the precipitation of chitosan.

A lower pH would not allow a short pseudo-thermogelation time.

According to the present invention, the hydroxylated base used forneutralization is preferably NaOH.

Inadequate stirring or too fast addition of aqueous hydroxylated baseinduce the precipitation of chitosan.

After complete addition of the hydroxylated base, thepseudo-thermosetting neutralized chitosan composition may be kept understirring for 10 minutes at temperature lower than 5° C.

The pseudo-thermosetting neutralized chitosan composition of the presentinvention can be stored at a temperature lower than 5° C. for up to 1month while gelation may progressively occur during longer storage.

When the temperature of the pseudo-thermosetting neutralized chitosancomposition is increased, for example after administration,pseudo-thermogelation occurs leading to the formation of aphosphate-free, transparent firm hydrogel. The higher is thetemperature, the shorter is the gelation time.

The process for preparing the pseudo-thermosetting neutralized chitosancomposition according to the present invention may further comprise, ifrequired, a step of sterilizing the homogeneously reacetylated chitosanbefore the step g) of solubilization. To obtain a sterile hydrogel, thepreparation is performed under aseptic conditions (e.g. under a laminarflow) and every added solution is previously filtered through a 0.22 μmfilter.

For example, sterilization may be performed by radiation or ideally bysteam sterilization of homogeneously reacetylated chitosan suspended inwater, as described by Yen (Yen S.F. et al., 2001, U.S. Pat. No.5,773,608).

If desired, the consistency of the phosphate-free, transparent chitosanhydrogel of the present invention may be increased by further addingbefore, during or after the solubilization, or before, during or afterneutralization of the chitosan solution, an appropriate amount of a diolhaving at least a distance of 4.7 Å between its hydroxyl groups.

Preferably, the diol added is 1,3-propanediol.

The amount of diol added will depend on the required degree ofconsistency of the hydrogel.

The gelation mechanism of the pseudo-thermosetting neutralized chitosancomposition of the present invention is driven by neutralization ofchitosan at low temperature. When temperature increases, the global pKaof chitosan decreases, which decreases its global charge density. Thisallows the formation of direct interactions (e.g. hydrogen bridges orhydrophobic interactions) between polymeric chains.

Therefore, gelation is not induced by a rise of temperature but isfavored by a rise of temperature, further decreasing the global chargedensity through a decrease of the global pKa of chitosan, leading topseudo-thermogelation.

In the prior art, using β-GP to neutralize chitosan appears only usefulto prevent the precipitation of non-reacetylated chitosan duringneutralization.

In the present invention, since homogeneously reacetylated chitosan issoluble enough to not precipitate at neutral pH, the addition of β-GP isnot required and neutralization may be advantageously performed with anyhydroxylated base such as NaOH.

In order to demonstrate the improved elastic properties of thephosphate-free, transparent chitosan hydrogels of the present invention,measurements of viscoelastic properties of two hydrogels according tothe invention and of one comparative hydrogel have been performedaccording to the following method.

Viscoelastic properties of hydrogels were determined immediately afterpreparation of the hydrogels using a Rheostress 1 (Haake, Karlsruhe,Germany) using a cone/plate device (diameter 60 mm, angle 4°).Temperature was controlled with a thermostatic bath Haake DC 30 and acooling device Haake K10 (Haake, Karlsruhe, Germany) coupled with therheometer. Hydrogels were placed between the cone and plate (cooled downat 4° C.) and measured after 10 minutes. All measurements were performedin the linear viscoelastic range and G′ and G″ were determined under aconstant deformation (γ=0.05) at 1.00 Hz as the temperature wasincreased from 4 to 37° C. at 1° C./min. over a period of 120 minutes.Temperature of the hydrogel was checked at the beginning and at the endof the measurement with a thermometer probe (RS-232, Extech Instruments,Waltham, USA). Evaporation of water leading to drying of hydrogels wasminimized by use of a cover surrounding the cone/plate device.

Hydrogels tested were:

-   -   the phosphate-free, transparent chitosan hydrogel of the present        invention obtained in Example 6, containing 0.75% of chitosan        (DD=48.6), neutralized at pH 7 by addition of NaOH;    -   the phosphate-free, transparent chitosan hydrogel of the present        invention obtained in Example 7, containing 0.75% of chitosan        (DD=48.6), added with 2.0 ml of 1,3-propanediol and neutralized        at pH 7 by addition of NaOH;    -   the comparative phosphate-containing, turbid chitosan hydrogel        obtained in Example 10 (Comparative), containing 0.75% of        chitosan (DD=83.2%) neutralized at pH 7 by addition of β-GP.

The elastic modulus (G′) of the tested hydrogels as a function of timewhen temperature increases from 4 to 37° C. are reported in the Figure.G″ is not represented to clarify the presentation of the Figure.

As shown by the Figure, the G′ values are much smaller in thecomparative chitosan hydrogel neutralized with β-GP than in the chitosanhydrogels neutralized with NaOH according to the present invention.

Further, as shown by the Figure, addition of a diol in the hydrogelaccording to the present invention further increases the G′ values ofthe hydrogel as compared with the same hydrogel not added with a diol.

These results show clearly that the viscoelastic properties of thephosphate-free, transparent chitosan hydrogels according to the presentinvention are increased as compared with phosphate-containing chitosanhydrogels known from the prior art.

Further, these results show that viscoelastic properties ofphosphate-free, transparent chitosan hydrogels according to the presentinvention are further increased by adding a diol.

The following examples are intended to illustrate the present invention.However, they cannot be considered in any case as limiting the scope ofthe present invention.

EXAMPLES

In the following examples, the deacetylation degree of chitosans wasdetermined by the spectrophotometric method described by R. A. Muzarelliand R. Richetti in “Chitin in Nature and Technology”, Plenum Press385-388, 1986 and the molecular weight of chitosans was determined bysize exclusion chromatography as reported by O. Felt, P. Furrer, J. M.Mayer, B. Plazonnet, P. Burri and R. Gurny in Int. J. Pharm. 180,185-193 (1999).

Further, in the following examples, the turbidity of hydrogels wasmeasured at 620 nm with a UV spectrophotometer using formazinsuspensions as standards. Briefly, hydrazine sulfate was reacted withhexamethylenetetramine to induce formazin precipitation. Standards ofknown formazin turbidity units (FTU) were prepared by appropriatedilution.

Example 1

Preparation of a Homogeneously Reacetylated Chitosan Having a DD of30.4%

The preparation of a homogeneously reacetylated chitosan having a DD of30.4% started from a chitosan of pharmaceutical grade having a DD of83.2% and a MW of 1′100 kDa (Aldrich Chemical, Milwaukee, USA).

10.0 g of chitosan were solubilized in 1000 ml of 10% aceticacid/methanol (20/80) mixture and successively filtered through 100 μmand 5 μm filters. Chitosan was precipitated by addition under stirringof 0.2 M NH₄OH/methanol (50/50) and washed 4 times with 3000 ml ofmethanol. Chitosan was dried for 3 days in the presence of Silicagel,under vacuum, at room temperature and protected from light. 1000.0 mg offiltrated chitosan were solubilized in 90.0 ml of 10% aceticacid/methanol (20/80) mixture. After complete dissolution, the solutionwas cooled down in an ice-bath. Reacetylation was performed in anice-bath. 360 μl of cold acetic anhydride were mixed in 10.0 ml of coldmethanol and this mixture was immediately added drop by drop to thechitosan solution under fast stirring. The solution was kept understirring for 1 h in the ice-bath and turned into a gel that was left atroom temperature for 7 h. This gel was transferred into dialysis bagsfor dialysis against 2 l deionized water for 1 week. The water waschanged twice a day. At the end of the purification step, the gel turnedinto a viscous solution, which was filtered through a 100 μm filter.

Reacetylated chitosan was precipitated by addition of 0.2 MNH₄OH/methanol (50/50) under stirring and washed 4 times with 300 ml ofmethanol. Reacetylated chitosan was dried for 3 days in the presence ofSilicagel, under vacuum, at room temperature and protected from light.At the end of the process, its DD was equal to 30.4%.

Example 2

Preparation of a Hydrogel from a Homogeneously Reacetylated ChitosanHaving a DD of 30.4%, Neutralization with NaOH

The hydrogel was prepared by the solubilization of 75.0 mg of chitosanobtained in Example 1 in a mixture of 1.0 ml of HCl 0.5N and 4.0 ml ofdeionized water. The solution was kept under stirring for 48 h at roomtemperature to allow complete chitosan solubilization. NaOH 0.1Npreviously cooled down in an ice-bath was added under stirring drop bydrop to adjust the pH to 7.0 in an ice-bath. The volume was completed to10.0 ml with water and the hydrogel was kept under stirring for 10minutes in an ice-bath. The phosphate-free hydrogel thus obtained wastransparent and had a turbidity of 3000 NTU at 37° C.

Example 3 (Comparative)

Preparation of a Homogeneously Reacetylated Chitosan Having a DD of64.9%

The preparation of a homogeneously reacetylated chitosan having a DD of64.9% started from a chitosan of pharmaceutical grade having a DD of83.2% and a MW of 1′100 kDa (Aldrich Chemical, Milwaukee, USA). It wasperformed as described in Example 1, except that the quantity of aceticanhydride was here equal to 200 μl. At the end of the process, its DDwas equal to 64.9%.

Example 4 (Comparative)

Preparation of a Hydrogel from a Homogeneously Reacetylated ChitosanHaving a DD of 64.9%, Neutralization with NaOH

The hydrogel containing a chitosan with a DD of 64.9% was prepared asdescribed in Example 2, except that the chitosan used was here thechitosan obtained in Example 3. The phosphate-free hydrogel obtained wasslightly turbid and had a turbidity of 9700 NTU at 37° C.

Example 5

Preparation of a Homogeneously Reacetylated Chitosan Having a DD of48.6%

The preparation of a homogeneously reacetylated chitosan having a DD of48.6% started from a chitosan of pharmaceutical grade having a DD of83.2% and a MW of 1′100 kDa (Aldrich Chemical, Milwaukee, USA). It wasperformed as described in Example 1, except that the quantity of aceticanhydride was here equal to 220 μl. At the end of the process, its DDwas equal to 48.6%.

Example 6

Preparation of a Hydrogel from a Homogeneously Reacetylated ChitosanHaving a DD of 48.6%, Neutralization with NaOH

75.0 mg of chitosan obtained in Example 5 were solubilized in a mixtureof 1.0 ml of HCl 0.5N and 4.0 ml of deionized water. The solution waskept under stirring for 48 h at room temperature to allow completechitosan solubilization. NaOH 0.1N previously cooled down in an ice-bathwas added under stirring drop by drop to adjust the pH to 7.0 in anice-bath. The volume was completed to 10.0 ml with water and thehydrogel was kept under stirring for10 minutes in an ice-bath. Thephosphate-free hydrogel thus obtained was transparent and had aturbidity of 2600 NTU at 37° C. Its viscoelastic behaviour followingtemperature increase is shown in the Figure.

Example 7

Preparation of a Hydrogel from a Homogeneously Reacetylated ChitosanHaving a DD of 48.6%, Neutralization with NaOH, Further Addition of aDiol

75.0 mg of chitosan obtained in Example 5 were solubilized in a mixtureof 1.0 ml of HCl 0.5N, 2.0 ml of deionized water and 2.0 ml of1,3-propanediol. The solution was kept under stirring for 48 h at roomtemperature to allow complete chitosan solubilization. NaOH previouslycooled down in an ice-bath 0.1N was added under stirring drop by drop toadjust the pH to 7.0 in an ice-bath. The volume was completed to 10.0 mlwith water and the hydrogel was kept under stirring for 10 minutes in anice-bath. The phosphate-free hydrogel thus obtained was transparent. Itsviscoelastic behaviour following temperature increase is shown in theFigure.

Example 8 (Comparative)

Preparation of a Non-Homogeneously Reacetylated Chitosan Having a DD of49.0%

The preparation of a non-homogeneously reacetylated chitosan having a DDof 49.0% started from a chitosan of pharmaceutical grade having a DD of83.2% and a MW of 1′100 kDa (Aldrich Chemical, Milwaukee, USA). It wasperformed as described in Example 1 for a homogeneously reacetylatedchitosan, but there was no previous filtration before reacetylation.Furthermore, reacetylation was performed at room temperature under lowstirring and acetic anhydride was not diluted in methanol beforeaddition. The quantity of acetic anhydride was here equal to 300 μl. Atthe end of the process, its DD was equal to 49.0%.

Example 9 (Comparative)

Preparation of a Hydrogel from a Non-Homogeneously Reacetylated ChitosanHaving a DD of 49.0%, Attempt of Neutralization with NaOH,Neutralization with β-GP

As the non-homogeneously reacetylated chitosan obtained in Example 8precipitated at neutral pH after neutralization with NaOH, a hydrogelwas prepared following the method described by Biosyntech. Consequently,neutralization was performed by addition of β-GP instead of NaOH.

75.0 mg of chitosan obtained in Example 8 (Comparative) were solubilizedin 5.0 ml of 0.1N HCl at room temperature for 48 h. After completedissolution, this solution was cooled down in an ice-bath. A solution of1000.0 mg of β-GP in 5.0 ml of deionised water was prepared at roomtemperature and cooled down in an ice-bath. The β-GP solution was addedto the chitosan solution under stirring drop by drop in an ice-bath,which adjusted the pH to 7.0. The hydrogel was kept under stirring for10 minutes in an ice-bath. The hydrogel thus obtained containedphosphate, was slightly turbid and had a turbidity of 4300 NTU at 37° C.

Example 10 (Comparative)

Preparation of a Hydrogel from a Commercial Chitosan Having a DD of83.2%. Neutralization with β-GP

75.0 mg of chitosan having a DD of 83.2% and a MW of 1′100 kDa (AldrichChemical, Milwaukee, USA) were solubilized in 5.0 ml of 0.1N HCl at roomtemperature for 48 h. After complete dissolution, this solution wascooled down in an ice-bath. A solution of 1000.0 mg of β-GP in 5.0 ml ofdeionized water was prepared at room temperature and cooled down in anice-bath. The β-GP solution was added to the chitosan solution understirring drop by drop in an ice-bath, which adjusted the pH to 7.0. Thehydrogel was kept under stirring for 10 minutes in an ice-bath. Thehydrogel thus obtained contained phosphate, was completely turbid andhad a turbidity of 15600 NTU at 37° C. Its viscoelastic propertiesfollowing temperature rise can be seen in the Figure.

Example 11 (Comparative)

Preparation of a Hydrogel from a Commercial Chitosan Having a DD of83.2% Attempt of Neutralization with NaOH

75.0 mg of chitosan having a DD of 83.2% and a MW of 1′100 kDa (AldrichChemical, Milwaukee, USA) were solubilized in 5.0 ml of 0.1N HCl at roomtemperature for 48 h. After complete dissolution, this solution wascooled down in an ice-bath. NaOH 0.1N was added under stirring drop bydrop, but after the addition of a few drops, chitosan started toprecipitate. It was therefore impossible to prepare a hydrogel.

1. A pseudo-thermosetting neutralized chitosan composition, whichcomprises 0.1 to 2.0 wt/v %, based on the total composition, of ahomogeneously reacetylated chitosan derived from a chitosan having adeacetylation degree of 80-90%, having a molecular weight of not smallerthan 200 kDa and a deacetylation degree of 30-60%, neutralized with anhydroxylated base, wherein said composition forms a phosphate-freetransparent hydrogel at a temperature higher than 5° C.
 2. Thepseudo-thermosetting neutralized chitosan composition according to claim1 , comprising 0.5 to 1 wt/v %, based on the total composition, of saidhomogeneously reacetylated chitosan.
 3. The pseudo-thermosettingneutralized chitosan composition according to claim 1, wherein thedeacetylation degree of said homogeneously reacetylated chitosan is 45to 55%.
 4. The pseudo-thermosetting neutralized chitosan compositionaccording to claim 1, wherein the molecular weight of said homogeneouslyreacetylated chitosan is not smaller than 600 kDa.
 5. Thepseudo-thermosetting neutralized chitosan composition according to claim1, further comprising a diol having a distance of at least 4.7 Å betweenits hydroxyl groups.
 6. The pseudo-thermosetting neutralized chitosancomposition according to claim 5, wherein said diol is 1,3-propanediol.7. A process for producing a homogeneously reacetylated chitosan havinga molecular weight of not smaller than 200 kDa and a deacetylationdegree of 30-60% which comprises the steps of: a) filtrating a chitosanhaving a molecular weight of not smaller than 200 kDa and adeacetylation degree of 80 to 90% dissolved in an acidic medium toeliminate insoluble particles; b) precipitating chitosan contained inthe filtrated acidic solution obtained in step a) to obtain chitosanfree of insoluble particles; c) preparing a cooled acidic solution ofthe chitosan free of insoluble particles obtained in step b) at atemperature lower than 5° C. to obtain a cooled acidic solution ofchitosan free of insoluble particles; d) preparing a cooled aceticanhydride solution containing a predetermined amount of acetic anhydridein methanol at a temperature lower than 5° C.; e) reacetylating chitosanby adding dropwise, under homogeneous conditions, the cooled aceticanhydride solution of step d) to the cooled solution of chitosanprepared in step c) to provide a crude homogeneously reacetylatedchitosan having a deacetylation degree of 30-60%; f) treating said crudechitosan obtained in step e) to eliminate salts produced duringreacetylation and insoluble particles of chitosan to obtain ahomogeneously reacetylated chitosan having a deacetylation degree of30-60%.
 8. The process according to claim 7, wherein the treating stepf) includes the steps of: f-1) dialyzing chitosan obtained in step e) toeliminate salts produced during reacetylation in order to obtain ahomogeneously reacetylated chitosan solution; f-2) filtrating thechitosan solution obtained in step f-1) to eliminate insoluble particlesof chitosan; f-3) precipitating chitosan contained in the filtratedsolution obtained in step f-2) and then drying chitosan to obtain ahomogeneously reacetylated chitosan having a deacetylation degree of30-60%.
 9. The process according to claim 8, wherein the precipitatingstep f-3) includes addition of a mixture of NH₄OH/methanol.
 10. Ahomogeneously reacetylated chitosan having a molecular weight of notsmaller than 200 kDa and a deacetylation degree of 30-60% obtained bythe process as claimed in claim 7 for use in the preparation of apseudo-thermosetting neutralized chitosan composition forming aphosphate-free transparent hydrogel at a temperature higher than 5° C.11. A process for producing a pseudo-thermosetting neutralized chitosancomposition forming a phosphate-free, transparent hydrogel at atemperature higher than 50° C., which comprises the steps of: g)solubilizing a homogeneously reacetylated chitosan derived from achitosan having a deacetylation degree of 80-90%, having a molecularweight of not smaller than 200 kDa and a deacetylation degree of 30-60%,in an aqueous HCl medium and cooling said acidic chitosan solution at atemperature lower than 5° C.; h) neutralizing the cooled chitosansolution obtained in step g) by adding an aqueous hydroxylated basepreviously cooled at a temperature lower than 5° C. to the cooledchitosan solution until the cooled solution of chitosan exhibits a pH of6.8-7.2; i) optionally, increasing the temperature of the neutral cooledsolution of chitosan obtained in step h) at a temperature higher than 5°C. in order to induce pseudo-thermogelation.
 12. The process accordingto claim 11, further comprising a step of sterilizing chitosan beforethe step g) of solubilization.
 13. The process according to claim 11,wherein in step h), the hydroxylated base is NaOH.
 14. The processaccording to claim 11, further comprising the step of adding anappropriate amount of a diol having a distance of at least 4.7 Å betweenthe hydroxyl groups before, during or after the solubilization step g),or before, during or after the neutralization step h) to increase theconsistency of the hydrogel to the required degree of consistency of thehydrogel.
 15. The process according to claim 14, wherein the diol is1,3-propanediol.
 16. The process according to claim 11, wherein thehomogeneously reacetylated chitosan solubilized in step g) is obtainedby: a) filtrating a chitosan having a molecular weight of not smallerthan 200 kDa and a deacetylation decree of 80 to 90% dissolved in anacidic medium to eliminate insoluble particles; b) precipitatingchitosan contained in the filtrated acidic solution obtained in step a)to obtain chitosan free of insoluble particles; c) preparing a cooledacidic solution of the chitosan free of insoluble particles obtained instep b) at a temperature lower than 5° C. to obtain a cooled acidicsolution of chitosan free of insoluble particles; d) preparing a cooledacetic anhydride solution containing a predetermined amount of aceticanhydride in methanol at a temperature lower than 5° C.; e)reacetylating chitosan by adding dropwise, under homogeneous conditions,the cooled acetic anhydride solution of step d) to the cooled solutionof chitosan prepared in step c) to provide a crude homogeneouslyreacetylated chitosan having a deacetylation degree of 30-60%; f)treating said crude chitosan obtained in step e) to eliminate saltsproduced during reacetylation and insoluble particles of chitosan toobtain a homogeneously reacetylated chitosan having a deacetylationdegree of 30-60%.
 17. A phosphate-free transparent pseudo-thermosettingchitosan hydrogel obtained by the process as claimed in claim
 11. 18. Ause of a homogeneously reacetylated chitosan having a deacetylationdegree of 30-60% and a molecular weight of not smaller than 200 kDaobtained by the process as claimed in claim 7, for the preparation of aphosphate-free, transparent, pseudo-thermosetting chitosan hydrogel. 19.A use of a pseudo-thermosetting neutralized chitosan composition asclaimed in claim 1, as a drug delivery system.
 20. The process accordingto claim 16, wherein the treating step f) includes the steps of; f-1)dialyzing chitosan obtained in step e) to eliminate salts producedduring reacetylation in order to obtain a homogeneously reacetylatedchitosan solution; f-2) filtrating the chitosan solution obtained instep f-1) to eliminate insoluble particles of chitosan; f-3)precipitating chitosan contained in the filtrated solution obtained instep f-2) and then drying chitosan to obtain a homogeneouslyreacetylated chitosan having a deacetylation degree of 30-60%.
 21. Theprocess according to claim 20, wherein the precipitating step f-3)includes addition of a mixture of NH₄OH/methanol.